Enhancement of a Two-Phase Partitioning Bioreactor System by Modi®cation of the Microbial Catalyst: Demonstration of Concept H. A. Vrionis, 1 A. M. Kropinski, 2 A. J. Daugulis 1 1 Department of Chemical Engineering, Queen's University, Kingston, Ontario K7L 3N6, Canada; telephone: 613-533-2784; fax: 613-533-6637; e-mail: daugulis@chee.queensu.ca 2 Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada Received 31 August 2001; accepted 21 February 2002 DOI: 10.1002/bit.10313 Abstract: Application of two-phase partitioning biore- actors (TPPB) to the degradation of phenol and xenobi- otics has been limited by the fact that many organic compounds that would otherwise be desirable delivery solvents can be utilized by the microorganisms em- ployed. The ability to metabolize the solvent itself could interfere with xenobiotic degradation, limiting remedia- tion ef®ciency, and hence represents a microbial char- acteristic incompatible with process goals. To avoid the issue of bioavailability, previous TPPB applications have relied on complex and often expensive delivery solvents or suboptimal catalyst±solvent pairings. In an effort to enhance TPPB activity and applicability, a genetically engineered derivative of Pseudomonas putida ATCC 11172 mutated in its ability to utilize medium-chain- length alcohols was generated (AVP2) and applied as the catalyst within a TPPB system with decanol as the de- livery solvent. Kinetic analysis veri®ed that the genetic alteration had not negatively affected phenol degrada- tion. The volumetric productivity of AVP2 (0.48 g/L h ±1 ) was equivalent to that seen for wild-type ATCC 11172 (0.51 g/L h ±1 ), but a comparison of initial cell concentra- tions and yields revealed an improved phenol-degrading ef®ciency for the mutant under process conditions. Yield coef®cients, cell dry weight, and viable count determi- nations all con®rmed the stability of the modi®ed phe- notype. This work illustrates the possibilities for TPPB process enhancement through a careful combination of genetic modi®cation and solvent selection. ã 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 587±594, 2002. Keywords: two-phase partitioning bioreactor; meta- bolic engineering; process enhancement; biodegrada- tion; solvent INTRODUCTION The release of various toxic compounds into the envi- ronment can often result in considerable costs to human health and the environment. The detrimental conse- quences of this release have included immediate toxicity to exposed biological populations, accumulation and degeneration of local sites, and pollutant transport leading to contamination of air, soil, groundwater, and waterways at locations far removed from the original site of discharge. Phenol is a toxic, highly water-soluble compound utilized in nylon production, and commonly found in the euents from coking and petroleum manufacturing plants. The extensive production and use of phenols in industry has resulted in wastewaters con- taining phenolic concentrations ranging as high as 3 g/L (Annadurai et al., 2000) and, consequently, phenol has been classi®ed as a priority pollutant by both Canadian and U.S. environmental protection organizations (Canadian Environmental Protection Agency, 2001; U.S. Environmental Protection Agency, 1998). Despite being toxic, various contaminants, including phenol, can be utilized by microbes as carbon and en- ergy sources (Gibson, 1968). Harnessing this degrada- tive potential has been an area of considerable study and numerous biodegradative remediation approaches have been developed with impressive success at eliminating xenobiotics from the environment (Garbisu and Al- korta, 1999). A key issue in biodegradative approaches to pollutant remediation is delivery of the toxic com- pound to the microbial catalysts at an appropriate concentration to avoid nutrient limitation while not exceeding the microbes' exposure threshold. This is a considerable challenge because contaminant levels in the environment are often several orders of magnitude greater than microbial tolerance and target environ- mental limits (Singleton, 1994). As a result, treatments of concentrated wastes through biodegradative ap- proaches have often been multistep processes requiring contaminant dilution prior to degradation. One strategy that has been developed to address issues of contaminant delivery is the two-phase partitioning bioreactor (TPPB) system (Collins and Daugulis, 1996). In this scheme, an immiscible and biocompatible organic solvent phase is used to deliver toxic substrates to cells Correspondence to: A. J. Daugulis ã 2002 Wiley Periodicals, Inc.